Elsevier

Neuroscience

Volume 108, Issue 1, 5 December 2001, Pages 69-81
Neuroscience

Dynamic localization and clustering of dendritic Kv2.1 voltage-dependent potassium channels in developing hippocampal neurons

https://doi.org/10.1016/S0306-4522(01)00476-6Get rights and content

Abstract

Dendritic excitability is modulated by the highly variable spatial and temporal expression pattern of voltage-dependent potassium channels. Somatodendritic Kv2.1 channels contribute a major component of delayed rectifier potassium current in cultured hippocampal neurons, where Kv2.1 is localized to large clusters on the soma and proximal dendrites. Here we found that dramatic differences exist in the clustering of endogenous Kv2.1 in cultured rat hippocampal GABAergic interneurons and glutamatergic pyramidal neurons. Studies on neurons developing in culture revealed that while a similar sequence of Kv2.1 localization and clustering occurred in both cell types, the process was temporally delayed in pyramidal cells. Localization and clustering of recombinant green fluorescent protein-tagged Kv2.1 occurred by the same sequence of events, and imaging of GFP–Kv2.1 clustering in living neurons revealed dynamic fusion events that underlie cluster formation. Overexpression of GFP–Kv2.1 accelerated the clustering program in pyramidal neurons such that the observed differences in Kv2.1 clustering in pyramidal neurons and interneurons were eliminated. Confocal imaging showed a preferential association of Kv2.1 with the basal membrane in cultured neurons, and electrophysiological recordings from neurons cultured on transistors revealed that Kv2.1 contributed the bulk of a previously described adherens junction delayed rectifier potassium conductance. Finally, Kv2.1 clusters were found spatially associated with ryanodine receptor intracellular Ca2+ ([Ca2+]i) release channels.

These findings reveal a stepwise assembly of Kv2.1 potassium channels into membrane clusters during development, and an association of these clusters with Ca2+ signaling apparatus. Together these data suggest that the restricted localization of Kv2.1 may play an important role in the previously observed contribution of this potassium channel in regulating dendritic [Ca2+]i transients.

Section snippets

Materials

Sprague–Dawley timed pregnant rats were purchased from Taconic Farms (Germantown, NY, USA). Horse serum was obtained from JRH Biosciences (Lenexa, KS, USA). Alexa 488 and Alexa 594 conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies were purchased from Molecular Probes (Eugene, OR, USA). Fluorescein 5-isothiocynate and Texas Red conjugated goat anti-mouse IgG1 and IgG2a secondary antibodies were purchased from Southern Biotechnology Associates (Atlanta, GA, USA).

Primary hippocampal neuronal cell cultures

Kv2.1 clustering differs between pyramidal neurons and interneurons

We showed previously that cultured hippocampal neurons express Kv2.1 potassium channels (Maletic-Savatic et al., 1995, Murakoshi and Trimmer, 1999). We double-stained embryonic hippocampal neurons after 14 days in vitro (14 DIV) culture with antibodies specific for Kv2.1 and for the neuronal marker microtubule-associated protein 2 (MAP2). This revealed that all of the neurons in the cultures exhibited robust Kv2.1 staining that was restricted to the soma and proximal dendrites. However, the

Discussion

Analyses of the expression and localization of both endogenous Kv2.1 and recombinant GFP–Kv2.1 in hippocampal neurons developing in culture revealed a stepwise progression of Kv2.1 localization. Kv2.1 expression is first detected in the ER, which is typical for the original biosynthetic pool of any plasma membrane protein. The ER is the site of subunit assembly, where for Kv channels four α subunits assemble into a channel protein complex (Papazian, 1999). Developmentally, staining for Kv2.1 in

Acknowledgements

The authors thank Joan Speh and Dr. Gail Mandel for use of the confocal microscope, and Drs. Matthew N. Rasband and Kenneth J. Rhodes for critically reviewing this manuscript. Supported by NIH Grants NS34375 and NS42225.

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